Journal of Environmental Quality 31:2120-2123 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
SHORT COMMUNICATIONS
Evaluation of Commercial Odor Control Agents for Suppressing Escherichia coli in Swine Manure Slurry
Della Johnstona,
Ralph Chapmana,
Daniel Masséb and
Edward Topp*,a
a Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V 4T3
b Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, P.O. Box 90, 2000 Route 108 East, Lennoxville, QC, Canada J1M 1Z3
* Corresponding author (toppe{at}agr.gc.ca)
Received for publication November 14, 2001.
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ABSTRACT
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Ten commercially available manure odor control agents were evaluated in bench-scale laboratory microcosms for their ability to inhibit or kill Escherichia coli, a commonly used indicator of fecal pollution and a potential pathogen. At manufacturer recommended rates, none of the agents reduced viable populations of E. coli in pure cultures or in swine manure slurry. However, at rates 10-fold higher than those recommended by the manufacturer, EnviroPur rapidly reduced viable populations of E. coli. Accelerated death of E. coli was observed at temperatures as low as 4°C. Chemical analysis of EnviroPur indicated that it contains alkylphenol polyethoxylates, common industrial surfactants. These results suggest that at manufacturer-recommended rates, the odor-controlling agents would not be effective at suppressing E. coli in stored swine manure slurry.
Abbreviations: TSB, trypticase soy broth
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INTRODUCTION
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IN MANY AREAS of Canada and elsewhere, livestock production is being intensified at the same time as agricultural areas are being urbanized. These developments are increasing concerns about water and air quality in proximity to livestock farms. In the swine industry, the common practice is to clean the barns and handle the animal wastes as a slurry, consisting of water (typically 90–95% v/v), feces, urine, and feed particles. A major environmental and social issue with the swine industry is the control of malodorous emissions from the slurry during storage and following land application. There are a number of commercial products that claim to limit odor from manure, some that are used as feed additives and some that are added to manure in the holding tanks (Zhu et al., 1997a,b; McCrory and Hobbs, 2001). Most of these products are found in one of the following categories: acidifying agents (acids, base precipitating salts, and substrates that induce acid production), adsorbents (clinoptilolite, peat), saponins (e.g., yucca [Yucca spp.] plant extract), masking agents (e.g., mixture of aromatic oils), digestive additives (selected microbial strains, enzymes), disinfectants (surfactants, chlorine, orthodichlorobenzene), and chemical oxidizing agents (H2O2, O3, KMnO4).
Some commercial odor control agents also claim to reduce or eliminate microbial pathogens present in swine manure slurry. Clearly, reducing the concentrations of potentially pathogenic bacteria in manure would be an attractive feature of an odor-controlling agent, reducing the risk to ground or surface water in proximity to manured fields from contamination with enteric bacteria. Escherichia coli is a commonly employed indicator of fecal pollution of water, and a number of strains of this bacterium possess virulence factors enabling them to cause disease (Donnenberg, 2000). The main objective of this study was to evaluate a number of commercially available odor control agents for their efficacy in destroying E. coli in manure slurry.
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Materials and Methods
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Microbiological Methods
The E. coli strain used for testing the inhibitory potential of the manure odor control agents was isolated from stored manure slurry from a finishing swine operation in southwestern Ontario, Canada. A sample was plated onto Endo Agar (Merck, Darmstadt, Germany), and a clone with the characteristic green metallic sheen was picked, purified on Endo Agar, and identified as E. coli with standard taxonomic tests (Gerhardt et al., 1994). The strain, designated Ec33, was routinely maintained on Luria–Bertoli agar (LBA). Cells to be used for inhibition assays were grown overnight in trypticase soy broth (TSB) at 44°C to an optical density (600 nm) of 1. Cells were harvested by centrifugation at 10 000 x g, and resuspended in sodium metaphosphate for addition to manure slurry. Inoculum densities were adjusted on the basis that an absorbance at 600 nm of 1 corresponded to a cell density of 1 x 109 colony forming units per milliliter (determined by plate counts on trypticase soy agar).
Escherichia coli was enumerated on either eosin methylene blue (EMB; Difco, Becton Dickinson, Sparks, MD) agar or Endo Agar. Enterococcus spp. were enumerated on m-enterococcus (Difco) agar. Samples of manure slurry were serially diluted in a dispersant consisting of 2 g sodium metaphosphate per liter for plating. Plates for E. coli enumeration were incubated for 24 h at 44°C, and for Enterococcus spp., 48 h at 37°C.
The potential E. coli growth inhibition by the odor-control agents was initially evaluated in small cultures. Ten milliliters of half-strength TSB in replicate 60-mL serum bottles were inoculated with E. coli to an initial cell density of 103 cells/mL. Odor control products were added at 1 or 10 times the manufacturer's recommended concentrations, and the serum bottles were sealed and incubated at 44°C with agitation. Samples were taken periodically for enumeration as described above.
Chemical Analyses
Manure samples were analyzed for chemical composition with standard methods (A & L Laboratories East, London, ON, Canada). The pH and redox potential were measured periodically during the incubations with an Accumet pH electrode (Orion Research, Beverly, MA) or a platinum redox electrode (Orion). Reported redox values are expressed as absolute millivolt values (i.e., all values are referenced to zero millivolts). The composition of EnviroPur was determined qualitatively with a Model LCT electrospray ionization mass spectrometer (Micromass, Manchester, UK) in the positive ion mode.
Manure Slurry Microcosms
The ability of odor control agents to influence the microbial composition of manure was evaluated in bench-scale microcosms. Manure slurry was collected from a manure holding tank on a 150-animal farrow-to-finish swine operation located near London, ON, Canada. The tank was open to the rain, and received barn washings. Upon sampling, the manure had 1.3% dry matter, of which 57% was lost upon ashing. The manure contained 0.23% total nitrogen and had a pH of 7.5. Portions of manure slurry (750 mL) were dispensed into 1-L mason jars, inoculated with 106 E. coli cells/mL slurry, and thoroughly mixed. Commercial odor-suppression products were added to duplicate microcosms at 1 and 10 times the manufacturer's recommended concentrations (Table 1). Microcosms were sealed with their lids, and incubated statically at 20°C for 8 to 15 d unless otherwise specified. Samples were periodically taken for plate count or other analyses.
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Table 1. Recommended rates of use of odor-control agents. Product characteristics and recommended rates are based on information accompanying the products.
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All treatments were in duplicate or triplicate, and data in figures are expressed as mean ± standard deviation. Statistically significant differences, considered to be at the P < 0.05 probability level, were established by subjecting data to a one-way analysis of variance (ANOVA) test.
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Results and Discussion
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When added to TSB cultures at recommended rates, there was no effect of any of the odor control agents on viable populations of E. coli (data not shown). However, when added at rates 10-fold higher than those recommended by the manufacturer, the EnviroPur product reduced viable populations of E. coli (Fig. 1)
. None of the products inhibited growth of E. coli to the maximum yield of 1010 colony forming units/mL, but EnviroPur greatly accelerated the decline in viable populations thereafter. After 72 h of incubation, a number of other products reduced the viable populations somewhat in comparison with untreated controls, but only marginally so. Likewise, there was no effect of any of the odor-control products on persistence of E. coli when added to manure slurry at concentrations recommended by the manufacturers for odor control (data not shown). However, when added to manure slurry at rates 10-fold higher than those recommended by the manufacturer, EnviroPur rapidly reduced E. coli populations to undetectable levels (Fig. 2)
. Several other products also somewhat accelerated the reduction in viable populations of E. coli following 8 d of incubation. At rates 10-fold higher than those recommended by the manufacturer, EnviroPur significantly hastened the reduction of viable populations of E. coli at temperatures ranging from 4 to 20°C compared with untreated controls incubated at comparable temperatures (Fig. 3)
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Fig. 1. Effect of manure odor control agents on viable populations of E. coli in half-strength trypticase soy broth (TSB). Agents were added at concentrations that were 10 times those recommended by the manufacturer.
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Fig. 2. Effect of manure odor control agents on viable populations of E. coli inoculated into manure slurry incubated statically at 20°C. Agents were added at concentrations that were 10 times those recommended by the manufacturer. The detection limit for the enumeration was 103 cells/mL.
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Fig. 3. Effect of manure odor control agents on persistence of E. coli inoculated in swine slurry incubated at 4, 12, or 20°C. Agents were added at concentrations that were 10 times those recommended by the manufacturer.
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For comparison, we enumerated populations of Enterococcus spp., indigenous to the manure during these experiments. These organisms are Gram positive indicators of fecal pollution. There was no detectable effect of any of these odor control products at rates 10-fold higher than recommended on viable populations of Enterococcus spp. (data not shown). This result indicates that bacteria in manure slurry will vary in their sensitivity to EnviroPur.
The odor control agents generally had little significant effect on the Eh or pH of the manure slurry. At the start of the incubations, the manure slurry microcosms (n = 22) had an Eh of -273 ± 5 mV and a pH of 7.5 ± 0.1. Following 24 d of incubation, the Eh was -387.6 ± 6.2, and pH 8.59 ± 0.05. The Eh in the EnviroPur-treated manure slurry at the end of the incubation (-400 ± 3 mV) was significantly lower than the mean across all other treatments. The basis for this small difference in redox potential due to EnviroPur is not known, but it is unlikely to be responsible for the observed result, particularly in light of EnviroPur's toxicity to E. coli in TSB.
Analysis by mass spectrometry revealed that EnviroPur contained a complex mixture of polyethoxylates based on C6, C7, and C8 alkyl phenols (alkyl phenol polyethoxylates, APEs) and on ethylene glycol monomethyl ether. The major use for APEs is as surfactants, important to a number of industrial applications including pulp and paper, textiles, coatings, agricultural pesticides, lube oils and fuels, metals, and plastics. The mixture probably contains alkylphenols, but this was not determined.
Octylphenol is weakly estrogenic, and it is considered an endocrine-disrupting substance (White et al., 1994). Xenoestrogenic alkylphenols such as octyl- or nonylphenol are products of the incomplete breakdown of APEs during anaerobic sewage treatment (Giger et al., 1984). Although 4-nonylphenol is rapidly degraded in soil (Topp and Starratt 2000), the disposition of alkylphenols during manure storage and following application of manure to soil may be an environmental consideration.
This study revealed that under laboratory conditions, EnviroPur odor control product applied at rates 10-fold higher than those recommended by the manufacturer could eliminate viable populations of E. coli in swine slurry at a range of temperatures that encompass those typical of manure storage lagoons. However, at the manufacturer's recommended concentrations, none of the manure odor control agents tested were effective in reducing viable populations of E. coli. The manure slurry we chose for this study had a solid matter content below that typically seen in slurries from many swine operations, and it is highly likely that at higher organic matter contents sorption will further reduce the effective concentration of these agents. These considerations reduce the likelihood that the manure control agents tested in this study will be effective at killing enteric bacteria at a reasonable cost, but further investigation at a commercial scale of manure storage is warranted.
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ACKNOWLEDGMENTS
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This work was partially funded by Ontario Pork, and Agriculture and Agri-Food Canada's Matching Investment Initiative. We thank Yuan-Ching Tien and Angela Dang for excellent technical assistance.
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REFERENCES
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ABSTRACT
INTRODUCTION
